Motile bacteria possess a stimulus-response system in which the input is the local concentration of a chemical attractant and the output is the motion of the cell. In Escherichia coli, the input is determined by the occupancy of a number of specific receptors, while the output is determined by the sense of rotation of about six flagellar filaments. When the filaments turn counter- clockwise, the cell swims steadily along a smooth path (it runs); when they turn clockwise, the cell moves in a highly erratic manner with little net displacement (it tumbles). Normally, these modes alternate, and the cell executes a three-dimensional random walk. The receptor occupancy over the past second is compared to the receptor occupancy over the previous three seconds, and the cell responds to the difference: runs are extended that carry the cell in a favorable direction. The present research focuses on the molecular events that couple the receptors to the flagella, i.e., the events required for chemotactic signaling. Four gene products are known to be involved, CheW, CheA, CheY and CheZ. CheY appears to be a second messenger that is activated by the receptors and diffuses to the flagella, where it enhances clockwise rotation (tumbles). Evidence from in vitro studies suggests that this activation results from phosphorylation, catalyzed by CheA. However, in vivo, CheY can be activated in the absence of CheA following the addition of acetate, via a pathway that appears to involve acetyl-CoA synthetase. Reconstitution experiments will be carried out in cells deleted for most of the chemotaxis genes to determine optimum stoichiometries for signaling for CheW, CheA and CheY, and to learn more about the kinetics of interaction of CheY, CheZ and the flagellar switch. Behavioral responses will be monitored in a tethered-cell assay. The acs gene will be clone and sequenced and its gene product characterized. Evidence for interaction with other components will be sought by a variety of means, including an in vitro search for acetylation or adenylylation. An in vitro motor assay will be developed that will allow rapid comparisons of the effects on switching of purified cytoplasmic components. Assays for judging the motility and chemotaxis of cell populations will be improved. Finally, the rotational behavior of tethered minicells will be analyzed to determine the switching statistics relevant when motors run at high speed. This will improve our understanding of the synchronization of different flagella on the same cell and allow more accurate predictions of swimming behavior based on the results of tethered-cell assays. The long-range goal is a description at the molecular level of sensory transduction in bacterial chemotaxis.